TY - JOUR
T1 - The 3S enantiomer drives enolase inhibitory activity in SF2312 and its analogues
AU - Pisaneschi, Federica
AU - Lin, Yu Hsi
AU - Leonard, Paul G.
AU - Satani, Nikunj
AU - Yan, Victoria C.
AU - Hammoudi, Naima
AU - Raghavan, Sudhir
AU - Link, Todd M.
AU - Georgiou, Dimitra K.
AU - Czako, Barbara
AU - Muller, Florian L.
N1 - Funding Information:
Funding: This research was funded by the NIH CDP SPORE P50CA127001-07 to F.L.M., The Research Scholar award, RSG-15-145-01-CDD from the American Cancer Society to F.L.M., The Young Investigator Award
Publisher Copyright:
© 2019 by the authors.
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2019
Y1 - 2019
N2 - We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel x-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.
AB - We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel x-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.
KW - Chiral
KW - E. coli
KW - Enolase
KW - Enzyme inhibitor
KW - Enzyme structure
KW - Glycolysis
KW - Hydroxamate
KW - Natural product
KW - Phosphonate
KW - X-ray crystallography
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U2 - 10.3390/molecules24132510
DO - 10.3390/molecules24132510
M3 - Article
C2 - 31324042
AN - SCOPUS:85068608284
VL - 24
JO - Molecules (Basel, Switzerland)
JF - Molecules (Basel, Switzerland)
SN - 1420-3049
IS - 13
M1 - , 2510
ER -